The present invention relates to a fluorescent microscope and a respective method for obtaining super-resolution images of a sample labelled with at least one type fluorescent label by combining localization microscopy and structured illumination microscopy.
The ability of a microscope to distinguishably image small, “point-like” objects is characterized, besides by the contrast, by the resolving power or resolution of the microscope. Due to the diffraction, point objects are seen as blurred disks (called Airy disks) surrounded by diffraction rings. The resolution of a microscope can be defined as the ability to distinguish between two closely spaced Airy disks. In other words, the resolution of a microscope can be defined as the ability to reveal adjacent structural details as distinct and separate. The diffraction limits this ability and thus the resolution. The extent and magnitude of the diffraction patterns are affected by both the wavelength of light λ and the numerical aperture (NA) of the objective lens. Thus, there is a finite limit beyond which it is impossible to resolve separate points in the object field, known as the diffraction limit or “Abbe/Rayleigh limit”.
Although the Abbe/Rayleigh limit is a universal principle that cannot be broken directly, multiple techniques, including fluorescence microscopy techniques, for surpassing it have been proposed. Contrary to normal transilluminated light microscopy, in fluorescence microscopy the observed sample (specimen or object) is illuminated through an objective lens with a narrow set of wavelengths of light. This light interacts with fluorophores in the sample which then emit light of a different wavelength, thereby forming an image of the observed specimen.
One technique to improve the resolution beyond the Abbe/Rayleigh limit is based on illumination of the sample with structured illumination light. Structured illumination microscopy (SIM or SMI) methods rely on coding high resolution information into the low resolution supported region of the microscope to circumvent the Abbe/Rayleigh limit. The required conditions are generated in structured illumination microscopy by illuminating the object with a periodic pattern and observing fluorescent light emitted by the illuminated sample. An example of a super-resolution fluorescent microscope using structured illumination light is disclosed in US 2010/0315708.
Another conceptually different technique to surpass the Abbe-Rayleigh limit based on Spectral Precision distance Microscopy (SPDM) or Localization microscopy (LM) is disclosed US 2009/115244 A1. The underlying principle of the SPDM/LM approach is the “optical isolation” in space and/or time domain and hence the independent localization of individual, “point-like” objects due to any photon-based characteristics of the emitted light. This means that in a given diffraction limited observation volume defined for example by the Full-Width-at-Half-Maxima (FWHM) of the Point Spread Function (PSF) of the microscope system used, at a given time interval and for a given spectral registration mode, only one point-like object (for example a single fluorescent or autofluorescent molecule) or (under certain conditions) only few objects are registered. By imaging fluorescent bursts of individual point-like objects (e.g. molecules) after excitation, the position of the point-like objects may be determined with a precision much higher than the FWHM of the PSF. The final super-resolution image is obtained by registration of multiple (up to thousands) of images of the same sample (or region of interest of a sample), so that the optical resolution is improved by “scanning” the fourth coordinate of the space-time continuum.
In fluorescent microscopes using structured illumination through an objective there is usually a substantial reduction of the intensity of the illumination light due to the employment of a system for generating structured illumination light. If the system for generating structured illumination light is an interferometric system (such as for example described in US 2010/0315708), there is a reduction of the initial light intensity of at least 50%. If diffraction gratings or spatial light modulators (SLM) are used to generate structured illumination light, the reduction in light intensity can be even higher.
For carrying out localization microscopic measurements, on the other hand, usually illumination light having high intensity is required, in order to assure sufficiently high photon yield. Thus for example, if the sample is labelled with “conventional” fluorophores, illumination light having high intensity in the range of about 10 kW/cm2 to about 1 MW/cm2 is needed in order to transfer the fluorescent molecules in a metastable dark state. If special photo-switchable fluorophores are used to label the sample, this initial illumination with high intensity light is not necessary. However, even in this case a read-out light with high intensity is needed, in order to sufficiently fast bleach out the individual activated fluorophores before new fluorophores are brought into the fluorescent state. An illumination light with low intensity results in a very long acquisition time. This problem is typical for many imaging fluorescent techniques.
In order to realise illumination of the sample with high intensity illumination light a high power laser is usually required. In addition, the size of illuminated region of the sample is usually very small. A reduction of the size of the illuminated region of the sample may be realized for example by an additional converging lens in the illumination path. Alternatively it is possible to use an optical set-up in which the laser light is not expanded, but is directly directed to the microscope objective instead. Still another option is to use a very low beam expansion, which is untypical for the fluorescent microscopy. In all cases the size of illuminated region of the sample in the localization microscopy is at best about 100 μm2. However, this reduces the amount of information acquired at each measurement. Thus, it is desirable to provide methods and apparatuses enabling the analysis of as large illuminated region as possible.
For the above reasons a combination of structured illumination microscopy and localization microscopy was considered very difficult, since either the intensity of the illumination light is too low for localization microscopy measurements or the size of the illuminated region of the sample is too small for structured illumination measurements.